Neutral polysaccharide wall coating for electrophoretic separations in capillaries and microchannels

ABSTRACT

The present invention describes a method of preparation of a neutral wall coating made of thermally immobilized polysaccharides. The coating suppresses electroosmotic flow and adsorption on the wall under acidic, neutral, and basic conditions in capillary electrophoresis.

TECHNICAL FIELD

The present invention generally relates to surface treatment, films and coatings, and more particularly coatings for chromatography and electrophoresis in capillaries and microchannels. Specifically, the invention is directed to wall coatings in capillary electrophoresis to reduce electroosmotic flow and adsorption of analytes on the wall.

BACKGROUND ART

Capillary electrophoresis has achieved a remarkably rapid development from its introduction in the early 1980s. This technique miniaturizes the electrophoretic process and presents significant advantages over traditional slab gel electrophoretic techniques. Most of materials used to prepare separation channels or capillaries for capillary electrophoresis (CE) contain ionizable groups on their surface that are responsible for so-called electrokinetic potential or ξ-potential. This potential is a cause of electroosmotic flow (EOF). The presence of EOF and especially its uneven distribution along the electrophoretic capillary or channel causes disturbances called eddy migration and a loss of resolution during electrophoretic separation. To eliminate EOF, a good wall coating eliminates ξ potential at the wall and/or increases viscosity inside the electric double layer. To reduce ξ-potential, it may react with charged groups incorporated in the wall (silanol groups in the case of fused silica capillary). To some extent, compounds with an opposite charge to the wall can be also used to titrate ξ-potential.

A number of various wall coatings have been proposed and developed to eliminate EOF and adsorption of analytes in fused silica capillaries. A vast majority of them merely reduced EOF and did not eliminate it completely. Frequently a dynamic wall coating was formed by simply adding an active ingredient to the background electrolyte. It adsorbs on the wall and reduces capillary surface charge and/or viscosity of solution in the electric double layer. Dynamic wall coatings are popular because of the simplicity of their preparation. However, they do not eliminate electroosmotic flow completely. Among many dynamic coatings, a guaran dynamic coating has been developed (Liu, Q., Lin, F., Hartwick, R. A. Capillary zone electrophoretic separation of basic proteins and drugs using guaran as a buffer modifier. Chromatographia 1998, 47, 219-224).

To eliminate EOF completely, static wall coatings have to be applied. Typically, a static wall coating is made of two layers: an intermediate layer and a hydrophilic polymer layer. A bifunctional reagent that reacts with both the capillary surface and functional groups of the polymer molecule usually forms the intermediate layer. The first polymer used for the preparation of a static wall coating was a linear polyacrylamide attached to the fused silica capillary wall by y-methacryloxypropyltrimethoxysilane (Hjertén, S., Coating for electrophoresis tube. U.S. Pat. No. 4,680,201). More hydrolytically stable coating was obtained when polyacrylamide was attached to the silica wall by using a Grignard reagent with an olephinic moiety, e.g., vinylmagnesium bromide after activating silanol groups by a reaction with thionyl chloride (Novotny, M. V.; Cobb, K. A., and Dolnik, V., Suppression of electroosmosis with hydrolytically stable coatings. U.S. Pat. No. 5,074,982; Novotny, M. V.; Cobb, K. A., and Dolnik, V., Suppression of electroosmosis with hydrolytically stable coatings. U.S. Pat. No. 5,143,753). Polyacrylamide is, however, hydrolytically unstable at high pH and hydrolyzes forming poly(acrylic acid). The presence of carboxylic groups leads to generation of ξ-potential on the wall and to an increase of EOF. A more stable wall coating is usually obtained if acrylamide is replaced with its derivative having some substituents on nitrogen (Dolnik, V. and Chiari, M., Compounds for molecular separations. U.S. Pat. No. 6,074,542).

Thermal immobilization of a polymer on a capillary wall is another way how to anchor a polymer on the capillary wall. Schomburg and coworkers proposed a poly(vinyl alcohol) (PVA) coating fixed thermally to the wall by heating the capillary at 140.degree.C. They assumed that formation of a permanent PVA coating was based on PVA becoming water insoluble by thermal treatment and expected PVA to form semicrystalline highly associated structures, which were not covalently bound to the fused silica capillary. PVA molecules became more strongly associated by hydrogen bridges and water molecules could not penetrate microcrystalline domains. The authors expressed their opinion that this was a unique property of PVA. In the pH range of 5-9, the PVA coating did not, however, completely eliminate EOF and the coated capillaries exhibited a pH-independent electroosmotic mobility of 1.2×10⁻⁹ m²V⁻¹s⁻¹ as measured in 20 mM sodium phosphate (Schomburg, G. and Gilges, M., Deactivation of the inner surfaces of capillaries. U.S. Pat. No. 5,502,169). The procedure was further modified to make a PVA wall coating on a glass microchip. A newly introduced crosslinking of PVA with glutaraldehyde should improve the stability of the coating. No heating is necessary, just drying is sufficient to provide a stable wall coating (Belder, D., Deege, A., Husmann, H., Kohler, F., and Ludwig, M. Cross-linked poly(vinyl alcohol) as permanent hydrophilic column coating for capillary electrophoresis. Electrophoresis. 2001; 22, 3813-3818).

Thermal immobilization was also applied to hydroxyethyl cellulose (HEC) and hydroxypropyl cellulose (HPC) (Shen, Y. and Smith, R. D. High-resolution capillary isoelectric focusing of proteins using highly hydrophilic-substituted cellulose-coated capillaries. J. Microcol. Sep. 2000; 12, 135-141). The authors found that the wall coating was stable if the silica capillaries were heated at 140° C. for 20 min rather than just being dried at room temperature for 4 days. From this observation they concluded that a chemical reaction must have occurred between cellulose derivatives and fused silica capillary inner wall.

There are several types of galactomannans, a class of linear polysaccharides with 1,4 linked β-D-mannopyranosyl units and 1,6-linked α-D-galactopyranosyl side groups (Dolnik, V., Gurske, W. A. and Padua, A.: Galactomannans as a sieving matrix in capillary electrophoresis. Electrophoresis 2001, 22, 707-719). The four most important galactomannans are locust bean gum, tara gum, guar gum (guaran), and fenugreek gum, which differ by the frequency of galactosyl side group attachment to the polymannose. The ratio of D-mannosyl to D-galactosyl units is approximately 3.8:1 for locust bean gum, 3:1 for tara gum, 1.8:1 for guar gum, and about 1:1 for fenugreek gum. Locust bean gum, guar gum, and tara gum are commercially produced and have various applications in the food industry and as an additive to fracturing fluids in the petroleum industry. Guar gum is obtained from the endosperm portion of the legume seed (Cyamopsis tetragonoloba) that grows mainly on the Indian subcontinent and in some parts of Texas and Oklahoma. Typical guar gum contains 75-85% of galactomannan, 8-14% water, 5-6% proteins, 2-3% fiber, and 0.5-1% ash. Guar gum shows an excellent resistance to shear degradation (Maier, H., Anderson, M., Karl, C., Magnus on, K., in: Whistler, R. L., BeMiller, J. N. (Eds.), Industrial Gums. Polysaccharides and Their Derivatives, Academic Press, San Diego 1993, pp. 181-226).

SUMMARY OF THE INVENTION

The present invention is useful as a capillary coating that satisfies the above objectives. The polysaccharide coating described here eliminates electroosmotic flow and reduces adsorption on the capillary wall. The coating is stable for at least 48 hours at pH 10.3.

The present invention is also a method of preparing said coating and using this coating to cover the interior of a capillary tube.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the idealized structure of guaran.

FIG. 2 shows the idealized structure of locust bean gum.

FIG. 3 demonstrates the effect of concentration of equimolar Tris-HEPES buffer on electroosmotic mobility of the coating.

FIG. 4 is an electropherogram of a model acidic protein mixture in guaran-coated capillary. Capillary: I(total)=335 mm, I(effective)=250 mm, ID=50 μm, OD=360 μm. BGE: 100 mM Tris-HEPES; voltage: −10 kV, injection: 30 mbar 3 s. Sample: 10 g/L polyglutamate, 4 g/L trypsin inhibitor, 8 g/L .alpha.-lactoglobulin, 1 g/L .beta.-lactalbumin.

FIG. 5 is an electropherogram of a model basic protein mixture in guaran-coated capillary. Capillary: I(total)=335 mm, I(effective)=250 mm, ID=75 μm, OD=360 μm. BGE: 100 mM .beta.-alanine-citric acid; voltage: −10 kV, injection: 30 mbar 3 s. Sample: 2 g/L polylysine, lysozyme, cytochrome c, trypsinogen, and .alpha.-lactalbumin.

FIG. 6 shows isoelectric focusing of synthetic pl standards in capillary coated with guaran. Capillary: I(total)=335 mm, I(effective)=250 mm, ID=75 μm, OD=360 μm. Anolyte: 20 mM citric acid, catholyte: 40 mM NaOH. Capillary filled with 1% Ampholines 3.5-10, 0.1% purified Synergel, 25 mM BisTris Propane. Focusing: 20 kV 8 min, mobilization: 100 mbar, 20 kV.

BEST MODE FOR CARRYING OUT THE INVENTION EXAMPLE 1

Preparation of thermally immobilized guaran wall coating

3 m piece of fused silica capillary 75 μm ID, 360 μm OD is flushed with 0.1 mL thionyl chloride under pressure of 500 psi to clean the inner surface of the capillary. Then the capillary is filled with 1 g/L guaran at pressure of 1000 psi. Guaran (FIG. 1) solution is prepared by dissolving purified guaran (Jaguar 2229, Rhodia, Hercules, Pa.) in deionized water and filtered through 0.2 μm nylon syringe filter (Crude guaran was previously purified by treatment with ion exchanger Source 30Q (Pharmacia, Uppsala, Sweden) and Amberlite MB-150 (Sigma, St. Louis, Mo.) followed by ion exchange treatment on Source 30S (Pharmacia, Uppsala, Sweden) and by precipitation with acetone (V. Dolnik, W. A. Gurske, and A. Padua: Solution of galactomannans as a sieving matrix in capillary electrophoresis. U.S. Patent Application 20020049184, Sep. 5, 2001).) The solution of guaran is left to flow through the capillary for 20 minutes. Then the capillary is flushed with nitrogen filtered through 0.2 μm nylon syringe filter at 1000 psi and after the capillary is emptied it is dried by flowing nitrogen at 100 psi through it. After a few minutes, the nitrogen flow is reduced to 20 psi and the capillary is placed in the oven heated to 102-105° C. for 30 minutes. After 30 minutes the capillary is cooled to room temperature and the nitrogen flow is stopped. The process can be repeated (without thionyl chloride treatment) providing multiple layer capillary coating.

Similarly other polysaccharides can be used to make a hydrophilic neutral wall coating including locust bean gum (FIG. 2), tara gum, fenugreek gum, scleroglucan, pullulan, and konjak, to name a few. In this way a hydrophilic film is prepared that may have other applications including homogenous lower layer for attachment of nucleic acid or proteins in preparation of microarrays.

EXAMPLE 2

Electroosmotic mobility of guaran wall coating and its dependence on electrolyte concentration

Electroosmotic mobility (μ_(EEO)) of the prepared wall coating is measured in a 335 mm long capillary with effective length of 250 mm using equimolar solution of tris(hydroxymethyl)aminomethane (Tris) and N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) as a background electrolyte (BGE) and 100 mM nicotinamide or 1 g/L hydroxyethyl methacrylate as a neutral marker. First a band of the marker is injected as a 2 s pulse at 50 mbar to the capillary inlet and is moved forward by pumping BGE electrolyte for 35 s at 50 mbar into capillary. Then another 2 s pulse of the neutral marker is injected at 50 mbar and moved forward by pumping BGE for 35 s at 50 mbar into capillary. The −10 kV voltage is applied for 180 s and a 3^(rd) pulse of the neutral marker (2 s at 50 mbar) is introduced into the capillary Then the all three bands of the neutral marker are pumped through the capillary by a pressure of 50 mbar, measuring absorption at 214 nm. Migration times of these three peaks are measured and used to calculate electroosmotic mobility (Williams, B. A. and Vigh, G., Determination of accurate electroosmotic mobility and analyte effective mobility values in the presence of charged interacting agents in capillary electrophoresis. Anal. Chem. 1997, 69, 4445-4451).

By using this method we measured electroosmotic mobility of the prepared capillaries. For high-quality coating that suppresses EOF significantly (μ_(EEO) below 10⁻⁹ m²V⁻¹s⁻¹), it is necessary to apply high voltage during measurement for extended period of time to keep reproducibility in an acceptable range.

The resulting electroosmotic mobility of the prepared coating depends on the ionic strength. The higher concentration of Tris-HEPES, the lower value of electroosmotic mobility was obtained (FIG. 3). Whereas in 1 mM Tris-HEPES the electroosmotic mobility typically exceeds 10⁻⁹ m²V¹s⁻¹, in 100 mM Tris-HEPES, the average values of μ_(EEO) are usually below 3×10^(−10 m) ²V⁻¹s⁻¹ and, sometimes they show a negative sign corresponding to reversed electroosmotic flow. This may be related to stronger interactions of Tris with the galactomannan at the wall than HEPES. This explanation is supported by the observation that background electrolytes containing certain anions, such as glutamate or TAPS, exhibit higher EOF than HEPES.

EXAMPLE 3

Capillary zone electrophoresis of acidic protein mixture

Quality of the prepared guaran wall coating was tested by CZE of model proteins in the guaran-coated capillary. The total length of the capillary was 335 mm, the effective length of the capillary was 250 mm. The capillary had ID 50 μm and OD 360 μm. For a CE separation of acid proteins, background electrolyte containing 100 mM Tris and 100 mM HEPES, pH 8.1 was used and a constant voltage of −10 kV was applied. The sample containing 10 g/L polyGlu, 4 g/L trypsin inhibitor, 8 g/L .beta.-lactoglobulin, 1 g/L .alpha.-lactalbumin in water was injected hydrodynamically applying pressure of 30 mbar for 3 s. The peaks were detected by measuring UV absorption at 214 nm. All five model proteins were separated in less than 25 minutes.

EXAMPLE 4

Capillary zone electrophoresis of basic protein mixture

Quality of the prepared guaran wall coating was tested by CZE of model proteins in the guaran-coated capillary. The total length of the capillary was 335 mm, the effective length of the capillary was 250 mm. The capillary had ID 75 μm and OD 360 μm. For a CE separation of acid proteins, background electrolyte containing 100 mM .beta.-alanine and 100 mM citric acid, pH 3.3 was used and a constant voltage of −10 kV was applied. The sample containing 2 g/L polylysine, lysozyme, cytochrome c, trypsinogen, and .alpha.-lactalbumin in water was injected hydrodynamically applying pressure of 30 mbar for 3 s. The peaks were detected by measuring UV absorption at 214 nm.

EXAMPLE 5

Capillary isoelectric focusing of pi standards in guaran-coated capillary

Utility of guaran-coated capillary for capillary isoelectric focusing was tested by isoelectric focusing of colored synthetic pl markers (FIG. 6). The capillary had ID 50 μm and OD 360 μm. For the focusing step, anolyte containing 20 mM citric acid and catholyte containing 40 mM NaOH were used. The capillary was filled with 1% Ampholines (pl range 3.5-10), 0.1% purified Synergel, and 25 mM BisTris Propane. In the focusing step 20 kV was applied for 8 min, then the focused zones were mobilized by applying pressure of 100 mbar, 20 kV. As the sample a mixture of five synthetic pi markers having pl 4.0, 5.3, 6.4, 7.5, 8.5 ({hacek over (S)}lais, K., Friedl, Z. Low-Molecular-Mass pl Markers for Isoelectric Focusing. Journal of Chromatography A 1994, 661, 249-256.) were injected. Migration times of the pl standards were linearly proportional to pl. In this particular case the relationship could have been expressed by the equation t _(m)=7.50+0.51 pl where t_(m) is migration time and pl isoelectric point.

REFERENCES CITED U.S. Patent Documents

Hjertén, S., Coating for electrophoresis tube. U.S. Pat. No. 4,680,201, 1987.

Novotny, M. V.; Cobb, K. A., and Dolnik, V., Suppression of electroosmosis with hydrolytically stable coatings. U.S. Pat. No. 5,074,982, 1990.

Novotny, M. V.; Cobb, K. A., and Dolnik, V., Suppression of electroosmosis with hydrolytically stable coatings. U.S. Pat. No. 5,143,753, 1991.

Dolnik, V. and Chiari, M., Compounds for molecular separations. U.S. Pat. No. 6,074,542, 2000.

Schomburg, G. and Gilges, M., Deactivation of the inner surfaces of capillaries. U.S. Pat. No. 5,502,169, 1996.

V. Dolnik, W. A. Gurske, and A. Padua: Solution of galactomannans as a sieving matrix in capillary electrophoresis. U.S. Patent Application 20020049184, Sep. 5, 2001.

Whistler R., Conversion of Guar Gum to Gel-Forming Polysaccharides by the Action of Alpha-Galactosidase. U.S. Pat. No. 4,332,894, 1982.

Other References

Maier, H., Anderson, M., Karl, C., Magnus on, K., in: Whistler, R. L., BeMiller, J. N. (Eds.), Industrial Gums. Polysaccharides and Their Derivatives, Academic Press, San Diego 1993, pp. 181-226.

Dolnik, V., Gurske, W. A. and Padua, A.: Galactomannans as a sieving matrix in capillary electrophoresis. Electrophoresis 2001, 22, 707-719.

Williams, B. A. and Vigh, G. Determination of accurate electroosmotic mobility and analyte effective mobility values in the presence of charged interacting agents in capillary electrophoresis. Anal. Chem. 1997. 69, 4445-4451.

Liu, Q., Lin, F., Hartwick, R. A. Capillary zone electrophoretic separation of basic proteins and drugs using guaran as a buffer modifier. Chromatographia 1998, 47, 219-224.

Belder, D. Deege, A., Husmann, H., Kohler, F., and Ludwig, M. Cross-linked poly(vinyl alcohol) as permanent hydrophilic column coating for capillary electrophoresis. Electrophoresis. 2001; 22, 3813-3818.

Shen, Y. and Smith, R. D. High-resolution capillary isoelectric focusing of proteins using highly hydrophilic-substituted cellulose-coated capillaries. J. Microcol Sep. 2000; 12, 135-141.

Slais, K., Friedl, Z. Low-Molecular-Mass pl Markers for Isoelectric Focusing. J. Chromatogr. A 1994, 661, 249-256. 

What is claimed is:
 1. A capillary system for electrophoretic separation of molecules, the system comprising: a separation column realized as a microchannel in a body or as a capillary, said separation column made of an electrically insulating material, said material comprising fused silica, glass, poly(methyl methacrylate), polycarbonate, poly(tetrafluoroethylene), cyclic polyolephines; a polymeric coating attached permanently to the interior surface of said separation column; electrode vials with electrodes in electrical connection with opposing ends of said separation column.
 2. A capillary system for electrophoretic separation of molecules of claim 1, comprising a polymeric coating attached permanently to the interior surface of said separation column, where the method of electrophoretic separation is capillary isoelectric focusing or capillary isotachophoresis.
 3. The coating of claim 1, wherein said coating comprises at least one permanent layer made of one or more of the following polysaccharides: locust bean gum, tara gum, guar gum, hydroxyisopropyl guaran, fenugreek gum, konjak, pullulan, pustullan, agarose, curdlan, laminaran, pustulan, dextran, tragacanth gum, amylose, schyzophyllan, nigeran, or scleroglucan.
 4. The coating of claim 3, wherein said polysaccharide is attached to the wall of said separation column by thermal immobilization.
 5. The coating of claim 4, wherein said thermal immobilization is performed by heating the separation column previously filled with a solution of said polysaccharide at temperature between 100 and 180° C. for 5-600 minutes.
 6. The coating of claim 5, wherein said thermal immobilization is performed in a protective atmosphere comprising at least 99.9 percent of the following dry gases: nitrogen, helium, or argon.
 7. The coating of claim 5, wherein said thermal immobilization is performed at a pressure 1 torr or less.
 8. The coating of claim 5, wherein said thermal immobilization is performed by heating the separation column in a protective atmosphere or under vacuum to temperature between 102 and 145° C. for 20-40 minutes.
 9. The coating of claim 5, wherein said polysaccharide is treated prior or after said thermal immobilization with an enzyme glycosidase that can cleave side saccharide groups and oligosaccharide branches from its polysaccharide backbone.
 10. The coating of claim 9, wherein said polysaccharide is locust bean gum, tara gum, guar gum, or fenugreek gum and said enzyme is α-galactosidase.
 11. The coating of claim 9, wherein said polysaccharide is amylose, dextran, nigeran, or pullulan and said enzyme is α-glucosidase.
 12. The coating of claim 9, wherein said polysaccharide is scleroglucan, schyzopyllan, pustulan, laminaran, or curdlan and said enzyme is β-glucosidase.
 13. The coating of claim 2, wherein said coating comprises at least one layer made of one or more of the following polysaccharides: locust bean gum, tara gum, guar gum, hydroxyisopropyl guaran, hydroxyethyl cellulose, fenugreek gum, konjak, pullulan, pustullan, agarose, curdlan, laminaran, pustulan, dextran, tragacanth gum, amylose, schyzophyllan, nigeran, or scleroglucan.
 14. The coating of claim 13, wherein said polysaccharide is attached to the wall of said separation channel be thermal immobilization.
 15. The coating of claim 14, wherein said thermal immobilization is performed by heating the separation channel previously filled with solution of said polysaccharide to temperature between 100 and 180° C. for 5-600 minutes.
 16. The coating of claim 15, wherein said thermal immobilization is performed in a protective atmosphere comprising at least 99.9 percent of dry gas: nitrogen, helium, or argon.
 17. The coating of claim 15, wherein said thermal immobilization is performed under pressure of 1 torr or less.
 18. The coating of claim 15, wherein said thermal immobilization is performed by heating of the separation channel in a protective atmosphere or under vacuum to temperature between 102 and 145° C. for 20-40 minutes.
 19. The coating of claim 13, wherein said polysaccharide is treated prior or after said thermal immobilization with an enzyme glycosidase that can cleave side saccharide groups and oligosaccharide branches from its polysaccharide backbone.
 20. The coating of claim 19, wherein said polysaccharide is locust bean gum, tara gum, guar gum, or fenugreek gum and said enzyme is α-galactosidase. 